Abstract
Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every PCR reaction product and assisting in choosing only the amplicons of interest with expected mutation. The principle of detection of small changes in DNA sequences is based on the changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence-dependent. The limitations faced in SSCP range from the routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems could be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP continues to remain the method of choice to analyze mutations and SNPs in order to understand genomic variations, spontaneous and induced, and the genetic basis of diseases.
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References
Graber, J. H., O’Donnell, M. J., Smith, C. L., and Cantor, C. R. (1998) Advances in DNA diagnostics. Curr. Opin. Biotechnol. 9, 14–18.
Lin, S. W., Lin, S. R., and Shen, M. C. (1993) Characterization of genetic defects of hemophilia A in patients of Chinese origin. Genomics 18, 496–504.
Pastor, P., Munoz, E., Ezquerra, M., et al. (2001) Analysis of the coding and the 5′ flanking regions of the α-synuclein gene in patients with Parkinson’s disease. Mov. Disord. 16, 1115–1119.
Higgins, J. J., Loveless, J. M., Goswami, S., et al. (2001) An atypical intronic deletion widens the spectrum of mutations in hereditary spastic paraplegia. Neurology 56, 1482–1485.
Chiavetto, L. B., Boin, F., Zanardini, R., et al. (2002) Association between promoter polymorphic haplotypes of interleukin-10 gene and schizophrenia. Biol. Psych. 51, 480–484.
Strippoli, P., Sarchielli, S., Santucci, R., Bagnara, G. N., Brandi, G., and Biasco, G. (2001) Cold single-strand conformation polymorphism analysis: optimization for detection of APC gene mutations in patients with familial adenomatous polyposis. Int. J. Mol. Med. 8, 567–572.
Suzui, M., Sugie, S., Mori, H., Okuno, M., Tanaka, T., and Moriwaki, H. (2001) Different mutation status of the β-catenin gene in carcinogen-induced colon, brain, and oral tumors in rats. Mol. Carcinogen. 32, 206–212.
French, J. E., Lacks, G. D., Trempus, C, et al. (2001) Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/−) mouse tumors is carcinogen-and tissue-dependent. Carcinogenesis 22, 99–106 [Erratum in Carcinogenesis 23, 373 (2002)].
Yamamoto, S., Tatematsu, M., Yamamoto, M., Fukami, H., and Fukushima, S. (1998) Clonal analysis of urothelial carcinomas in C3H/HeN↔BALB/c chimeric mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine. Carcinogenesis 19, 855–860.
Devereux, T. R., Anna, C. H., Foley, J. F., White, C. M., Sills, R. C, and Barrett, J. C. (1999) Mutation of β-catenin is an early event in chemically induced mouse hepatocellular carcinogenesis. Oncogene 18, 4726–4733.
Wanner, R., Zober, A., Abraham, K., Kleffe, J., Henz, B. M., and Wittig, B. (1999) Polymorphism at codon 554 of the human Ah receptor: different allelic frequencies in Caucasians and Japanese and no correlation with severity of TCDD induced chloracne in chemical workers. Pharmacogenetics 9, 777–780.
Hsu, C. H., Yang, S. A., Wang, J. Y., Yu, H. S., and Lin, S. R. (1999) Mutational spectrum of p53 gene in arsenic-related skin cancers from the blackfoot disease endemic area of Taiwan. Br. J. Cancer 80, 1080–1086.
Sarkar, F. H., Li, Y., and Vallyathan, V. (2001) Molecular analysis of p53 and K-ras in lung carcinomas of coal miners. Int. J. Mol. Med. 8, 453–459.
Nohturfft, A., Hua, X., Brown, M. S., and Goldstein, J. L. (1996) Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines. Proc. Natl. Acad. Sci. USA 93, 13709–13714.
Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86, 2766–2770.
Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.
Oto, M., Miyake, S., and Yuasa, Y. (1993) Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus. Anal. Biochem. 213, 19–22.
Iwahana, H., Yoshimoto, K., Mizusawa, N., Kudo, E., and Itakura, M. (1999) Multiple fluorescence-based PCR-SSCP analysis. BioTechniques 27, 20–22, 24.
Kukita, Y., Tahira, T., Sommer, S. S., and Hayashi, K. (1997) SSCP analysis of long DNA fragments in low pH gel. Hum. Mutat. 10, 400–407.
Glavac, D. and Dean, M. (1993) Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations. Hum. Mutat. 2, 404–414.
Yip, S. P., Hopkinson, D. A., and Whitehouse, D. B. (1999) Improvement of SSCP analysis by use of denaturants. BioTechniques 27, 20–22, 24.
**e, T., Ho, S. L., and Ma, O. C. (1997) High resolution single strand conformation polymorphism analysis using formamide and ethidium bromide staining. Mol. Pathol. 50, 276–278.
Markoff, A., Savoa, A., Vladimirov, V., Bogdanova, N., Kremensky, I., and Ganev, V. (1997) Optimization of single-strand conformation polymorphism analysis in the presence of polyethylene glycol. Clin. Chem. 43, 30–33.
Ushijima, T., Hosoya, Y., Suzuki, T., Sofuni, T., Sugimura, T., and Nagao, M. (1994) A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity. Nucleic Acids Res. 22, 2155–2157.
Savov, A., Angelicheva, D., Jordanova, A., Eigel, A., and Kalaydjieva, L. (1992) High percentage acrylamide gels improve resolution in SSCP analysis. Nucleic Acids Res. 20, 6741–6742.
Lallas, T. A. and Buller, R. E. (1998) Optimization of PCR and electrophoresis conditions enhances mutation analysis of the BRCA1 gene. Mol. Genet. Metab. 64, 173–176.
Ravnik-Glavac, M., Glavac, D., and Dean, M. (1994) Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum. Mol. Genet. 3, 801–807.
Vidal-Puig, A. and Moller, D. E. (1994) Comparative sensitivity of alternative single-strand conformation polymorphism (SSCP) methods. BioTechniques 17, 490–492, 494, 496.
Mohabeer, A. J., Hiti, A. L., and Martin, W. J. (1991) Non-radioactive single strand conformation polymorphism (SSCP) using the Pharmacia PhastSystem. Nucleic Acids Res. 19, 3154.
Monckton, D. G. and Jeffreys, A. J. (1994) Minisatellite isoalleles can be distinguished by single-stranded conformational polymorphism analysis in agarose gels. Nucleic Acids Res. 22, 2155–2157.
Liu, Q., Li, X., and Sommer, S. S. (1999) pK-matched running buffers for gel electrophoresis. Anal. Biochem. 270, 112–122.
Kaczanowski, R., Trzeciak, L., and Kucharczyk, K. (2001) Multitemperature single-strand conformation polymorphism. Electrophoresis 22, 3539–3545.
Hongyo, T., Buzard, G. S., Calvert, R. J., and Weghorst, C. M. (1993) ‘Cold SSCP’: a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses. Nucleic Acids Res. 21, 3637–3642.
Kozlowski, P. and Krzyzosiak, W. J. (2001) Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection. Nucleic Acids Res. 29, E71.
Hayashi, K. (1992) PCR-SSCP: a method for detection of mutations. Genet. Anal. Tech. Appl. 9, 73–79.
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Gupta, V. et al. (2005). Gel-Based Nonradioactive Single-Strand Conformational Polymorphism and Mutation Detection. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:247
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DOI: https://doi.org/10.1385/1-59259-840-4:247
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